Methods for sequencing a polynucleotide template

ABSTRACT

The invention relates to methods for pairwise sequencing of a polynucleotide template which result in the sequential determination of nucleotide sequence in two distinct and separate regions of the polynucleotide template.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation application of U.S. patent application Ser. No.11/989,170, filed Aug. 10, 2010, which is the § 371 U.S. National StageApplication of International Application No. PCT/GB2006/002709, filedJul. 20, 2006, which claims the benefit of Great Britain ApplicationSerial No. 0514935.6, filed Jul. 20, 2005, each of which is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to methods for pairwise sequencing of apolynucleotide template, which methods result in the sequentialdetermination of nucleotide sequence in two distinct and separateregions of the polynucleotide template.

BACKGROUND TO THE INVENTION

Advances in the study of biological molecules have been led, in part, byimprovement in technologies used to characterise the molecules or theirbiological reactions. In particular, the study of the nucleic acids DNAand RNA has benefited from developing technologies used for sequenceanalysis.

U.S. Pat. No. 5,302,509 describes a method for sequencing apolynucleotide template which involves performing multiple extensionreactions using a DNA polymerase or DNA ligase to successivelyincorporate labelled polynucleotides complementary to a template strand.In such a “sequencing by synthesis” reaction a new polynucleotide strandbased-paired to the template strand is built up in the 5′ to 3′direction by successive incorporation of individual nucleotidescomplementary to the template strand. The substrate nucleosidetriphosphates used in the sequencing reaction are labelled at the 3′position with different 3′ labels, permitting determination of theidentity of the incorporated nucleotide as successive nucleotides areadded.

In order to carry out accurate sequencing a reversible chain-terminatingstructural modification or “blocking group” may be added to thesubstrate nucleosides to ensure that nucleotides are incorporated one ata time in a controlled manner. As each single nucleotide isincorporated, the blocking group prevents any further nucleotideincorporation into the polynucleotide chain. Once the identity of thelast-incorporated labelled nucleotide has been determined the labelmoiety and blocking group are removed, allowing the next blocked,labelled nucleotide to be incorporated in a subsequent round ofsequencing.

In certain circumstances the amount of sequence data that can bereliably obtained with the use of sequencing-by-synthesis techniques,particularly when using blocked, labelled nucleotides, may be limited,typically to around 25-30 cycles of incorporation. Whilst sequencing“runs” of this length are extremely useful, particularly in applicationssuch as, for example, SNP analysis and genotyping, it would beadvantageous in many circumstances to be able to reliably obtain furthersequence data for the same template molecule.

The technique of “paired-end” or “pairwise” sequencing is generallyknown in the art of molecular biology, particularly in the context ofwhole-genomic shotgun sequencing (Siegel A. F. et al., Genomics. 2000,68: 237-246; Roach J. C. et al., Genomics. 1995, 26: 345-353).Paired-end sequencing allows the determination of two “reads” ofsequence from two places on a single polynucleotide template. Theadvantage of the paired-end approach is that there is significantly moreinformation to be gained from sequencing two stretches each of “n” basesfrom a single template than from sequencing “n” bases from each of twoindependent templates in a random fashion. With the use of appropriatesoftware tools for the assembly of sequence information (Millikin S. C.et al., Genome Res. 2003, 13: 81-90; Kent, W. J. et al., Genome Res.2001, 11: 1541-8) it is possible to make use of the knowledge that the“paired-end” sequences are not completely random, but are known to occuron a single template, and are therefore very close in the genome. Thisinformation has been shown to greatly aid the assembly of whole genomesequence into a consensus sequence.

Paired-end sequencing has typically been performed by making use ofspecialized circular shotgun cloning vectors known in the art. Aftercutting the vector at a specific single site, the template DNA to besequenced (typically genomic DNA) is inserted into the vector and theends resealed to form a new construct. The vector sequences flanking theinsert DNA include binding sites for sequencing primers which permitsequencing of the insert DNA on opposite strands.

A disadvantage of this approach is that it requires time-consumingcloning of the DNA templates it is desired to sequence into anappropriate sequencing vector.

Furthermore, because of the need to clone the DNA template into a vectorin order to position binding sites for sequencing primers at both endsof the template fragment it is extremely difficult to make use ofarray-based sequencing techniques. With array-based techniques it isgenerally only possible to sequence from one end of a nucleotidetemplate, this often being the end proximal to the point of attachmentto the array.

With the use of hairpin nucleic acid anchors or double stranded nucleicacid anchors (such as those described in the applicant's co-pendingInternational application published as WO 01/57248), one end of atemplate immobilised on an array may be “covalently closed” giving afree 3′ end which permits sequencing of the 5′ overhanging templatestrand by successive incorporation of nucleotides. However, given thatthe distal portions (distal from the point of attachment to the array)of such immobilised templates are generally single-stranded and that thesequence of the template is usually unknown prior to immobilisation onthe array, it is not straightforward to devise means for determining thesequence of the distal end of the immobilised template, beyond the first“run” of sequence that can be obtained from the free 3′ end provided bythe anchor. It is not possible simply to design a sequencing primercomplementary to a region of the template whose sequence is unknown.

WO 2004/070005 describes a method for double-ended sequencing of apolynucleotide template which can be carried out on a solid support. Themethod relies on simultaneous hybridisation of two or more primers to atarget polynucleotide in a single primer hybridization step. Followingthe hybridization step, all of the primers hybridized to the templateare blocked except for one, which has a free 3′ hydroxyl group whichserves as an initiation point for a first sequencing reaction.Sequencing proceeds until no further chain elongation is possible, orelse the sequencing reaction is terminated. Then one of the blockedprimers is unblocked to give a free 3′ hydroxyl and a second sequencingreaction is performed from this initiation point. An advantage of thisapproach is that there is no need to perform any denaturation andre-hybridization steps between the first and second sequencingreactions, as the two primers providing the initiation points areannealed in a single hybridisation step. Thus, the template remainsintact and attached to the solid support throughout.

A major drawback of this approach based in hybridisation of blocked andunblocked primers is that it is necessary to know the sequence of atleast two regions of the polynucleotide template to be sequenced inorder to design two or more suitable primers capable of binding to thetarget. If the method is to be used to sequence polynucleotides ofunknown sequence then it is necessary to carry out sample preparationsteps in order to add regions of known sequence to the polynucleotide tobe sequenced in order to provide the necessary primer-binding sites.This can be achieved, for example, by amplification or by sub-cloning atemplate of unknown sequence into a vector in order to add knownsequences onto the 5′ and 3′ ends of the template.

The present inventors have sought to develop techniques which generallypermit the paired-end or pairwise sequencing approach to be used withoutany knowledge of the sequence at the distal end of the template andwithout the need for any intermediate cloning of the template into avector. Such techniques would permit pairwise sequencing to be used inconjunction with a wide range of array-based sequencing technologies,including single molecule arrays as well as clustered arrays.

DESCRIPTION OF THE INVENTION

In accordance with a first aspect of the invention there is provided amethod for pairwise sequencing of a polynucleotide template, the methodcomprising:

(a) providing a polynucleotide template having a first free 3′ hydroxylgroup which is positioned to initiate sequencing of a first region forsequence determination on a first template strand,(b) carrying out a first sequencing reaction by sequential addition ofnucleotides to the first free 3′ hydroxyl group to determine thesequence of the first template strand in the first region,(c) generating a second free 3′ hydroxyl group within the polynucleotidetemplate at a nucleotide position which is spaced apart from the lastnucleotide added in the sequencing reaction of step (b), wherein thesecond free 3′ hydroxyl group is positioned to initiate sequencing of asecond region for sequence determination on a second template strand,and wherein the second free hydroxyl group is in continuous covalentlinkage with the last nucleotide added in the sequencing reaction ofstep (b), and(d) carrying out a second sequencing reaction by sequential addition ofnucleotides to the second free 3′ hydroxyl group to determine thesequence of the second template strand in the second region.

The invention provides a method for sequencing two regions of apolynucleotide template, referred to herein as the first and secondregions for sequence determination. The first and second regions forsequence determination may be on the same strand of a single-strandedpolynucleotide template, or they may be on complementary strands of adouble-stranded or self-complementary polynucleotide template.

If the first and second regions for sequence determination occur on asingle polynucleotide strand then they must not be immediately adjacentto each other, meaning that the last nucleotide to be sequenced in thefirst region is not in phosphodiester linkage with the first nucleotideto be sequenced in the second region. It is an essential feature thatthe two regions for sequence determination must be separated by at leastone nucleotide, and are preferably separated by at least 50, morepreferably at least 100 or more preferably at least 500 nucleotides.

If the first and second regions for sequence determination occur oncomplementary strands of a double-stranded polynucleotide template, or aself-complementary template, then the two regions may or may not becomplementary to each other.

A key feature of the method of the invention is that is does not requireprimer hybridisation to the template to be sequenced in order to provideinitiation points for the first and second sequencing reactions. This isa particular advantage when the method is carried out on a solidsupport, such as a chip-based array, since there is no need to carry outany hybridisation reactions on the solid support.

The starting point for the method of the invention is the provision of apolynucleotide template including a first free 3′ hydroxyl group whichcan serve as an initiation point for a sequencing reaction.

In the context of this invention the term “sequencing reaction” refersto any polynucleotide “sequencing-by-synthesis” reaction which involvessequential addition of nucleotides to a growing polynucleotide chain inthe 5′ to 3′ direction using a polymerase in order to form an extendedpolynucleotide chain complementary to the template region to besequenced. The identity of the added nucleotide is preferably determinedafter each addition step, thus the sequence of the template may beinferred using conventional Watson-Crick base-pairing rules.

The starting polynucleotide template is at least partiallydouble-stranded, or self-complementary. The first free 3′ hydroxyl groupis provided on one strand that is base-paired to a complementary strand(which can be the same polynucleotide strand if the template isself-complementary), which forms the first strand of the template to besequenced (first template strand), in the region immediately upstream ofthe first 3′hydroxyl group. The region of base-pairing between thestrand bearing the first hydroxyl group and the first template strandneed only be long enough to hold the two strands together under theconditions used for the subsequent sequencing reaction, in order thatthe hydroxyl group is positioned to initiate the sequencing reaction.Therefore, this region of base-pairing may be as short as from 10 to 13base-pairs. In embodiments wherein the template is linked to a hairpinlinker (see below), the region of base-pairing is provided by the “stem”portion of the hairpin linker.

The first template strand overhangs the first 3′ hydroxyl group in orderto define a first region of the template to be sequenced. Theoverhanging portion of the first template strand may be single-stranded.

Alternatively, the overhanging region may be annealed to a complementarystrand downstream of the free 3′ hydroxyl. The latter arrangement may beused if the first sequencing reaction is to be performed using astrand-displacement polymerase. The length of the overhanging strand maybe from 100 nucleotides (or base-pairs if double-stranded) up to 1 kb,or even longer. Preferably the overhanging strand will be at least 500nucleotide (or base-pairs).

The template may be provided as a single polynucleotide molecule whichis partially self-complementary. For example, the templatepolynucleotide molecule may fold back on itself to form aself-complementary hairpin structure at its 3′ end.

In one embodiment the starting polynucleotide may be formed by ligatinga self-complementary hairpin linker polynucleotide to the desiredpolynucleotide template to be sequenced, such that the 5′ end of thehairpin linker is joined to the 3′ end of the first template strand. Thefirst free 3′ hydroxyl group is then provided by the 3′ end of thehairpin linker. An advantage of this approach is that it may be used forsequencing templates of unknown sequence. Suitable self-complementaryhairpin polynucleotide linkers include those described in theapplicant's published International application WO 01/57258.

A first sequencing reaction is carried out by sequential addition ofnucleotides to the first free 3′ hydroxyl group. The nature of the addednucleotide is preferably determined after each nucleotide addition inorder to determined the sequence of the first template strand in thefirst region for sequence determination. This first sequencing reactioncan proceed for as long as is technically possible within thelimitations of the chosen sequencing methodology, for example until thesequence obtained is no longer accurate or reliable. The firstsequencing reaction will preferably involve sequencing of from 10 to 200or more consecutive bases, preferably from 15 to 35 consecutive bases ofthe first template strand, preferably from 20 to 30 consecutive basesand more preferably about 25 consecutive bases of the first templatestrand. The precise number of bases sequenced will be dependent upon thelimitations of the chosen sequencing methodology, and more specificallythe methodology used to determine the nature of the base added in eachnucleotide addition step.

Once the first sequencing reaction is deemed to be complete, sequencingof a second region of the polynucleotide template can be carried out. Inorder to sequence a second region of the template it is necessary togenerate a second free 3′ hydroxyl group which serves as an initiationpoint for a second sequencing reaction. The second free 3′ hydroxylgroup must be provided on a polynucleotide strand which is base-pairedto the second template strand to be sequenced in the region immediatelyupstream of the second free 3′ hydroxyl group. The second templatestrand overhangs the second free 3′ hydroxyl group to define a secondregion of the template to be sequenced. The first and second templatestrands may be the same strand of the polynucleotide template if thetemplate is single-stranded, or self-complementary.

It is an essential feature of the invention that the second free 3′hydroxyl group is “spaced apart from” the last nucleotide added in thefirst sequencing reaction, meaning that the second free 3′ hydroxylgroup must not be provided by the last nucleotide added in the firstsequencing reaction, and that there must be at least one nucleotideseparating the last nucleotide added in the first sequencing reactionand the nucleotide which provides the second free 3′ hydroxyl group.Preferably the last nucleotide added in the first sequencing reactionand the nucleotide which provides the second free 3′ hydroxyl group willbe separated by at least 50 nucleotides, more preferably at least 100nucleotides and more preferably at least 500 nucleotides.

It is also a feature of the invention that the last nucleotide added inthe first sequencing reaction and the nucleotide which provides thesecond free 3′ hydroxyl group should be generated in “continuouscovalent linkage”. This means that the last nucleotide added in thefirst sequencing reaction and the nucleotide which provides the secondfree 3′ hydroxyl group should form part of a single polynucleotidemolecule with continuous backbone phosphodiester linkage, or at leastthat they should have been so-joined on a single polynucleotide moleculeat some stage in the sequencing method. This polynucleotide molecule maybe a single strand or it may be a partially self-complementarypolynucleotide strand, having a hairpin or stem-loop structure.

The requirement for the last nucleotide added in the first sequencingreaction and the nucleotide which provides the second free 3′ hydroxylgroup to be in continuous covalent linkage is advantageous for pairwisesequencing of templates of unknown sequence, since it overcomes theproblem of designing a primer to initiate sequencing of second region ofa template of unknown sequence. Thus, the second free 3′ hydroxyl groupis NOT provided by hybridisation of a separate primer. This in turnmeans that when the method of the invention is carried out on an arraythere is no need to carry out any hybridisation steps after thetemplates are immobilised on the array.

There are several different ways in which the second free 3′ hydroxylgroup may be generated, as will be discussed in further detail below.

Non-limiting embodiments of the invention are now described in detailwith reference to the accompanying drawings, in which:

FIGS. 1 and 2 illustrate different embodiments of the sequencing methodaccording to the invention which allow sequencing of first and secondregions of a single-stranded polynucleotide template;

FIGS. 3 to 6 illustrate different embodiments of the sequencing methodaccording to the invention which allow sequencing of first and secondregions on complementary strands of a double-stranded polynucleotidetemplate.

Referring to FIG. 1A, a polynucleotide template (1) is provided, havinga first free 3′ hydroxyl group (2). In this embodiment the template tobe sequenced is a single polynucleotide strand (3). In the embodimentshown in FIG. 1A the first free 3′ hydroxyl group is provided by the 3′end of a self-complementary hairpin linker (4) which is ligated to thetemplate strand (3) such that the 5′ end of the linker (4) is joined tothe 3′ end of the template strand (3). The linker is also linked to asolid support (5) via any suitable linkage (6). The template strand (3)overhangs the first free 3′ hydroxyl group to define a first region ofthe template to be sequenced (7). Although the specific embodiment shownin FIG. 1A involves use of a hairpin which acts as a primer forsequencing and provides a means for attachment to a solid support theinvention is not intended to be limited to the use of hairpins. Anysuitable oligonucleotide primer hybridised to the template strand (3)could be used to provide an initiation point for sequencing. Linkage tothe solid support could be provided via the primer or via the templatestrand itself. If linkage to the solid support is provided via thetemplate strand then this may occur at the 5′ or the 3′ end of thetemplate strand, or even via an internal portion of the templateprovided that this does not interfere with subsequent sequencingreactions. Template strands generated by solid-phase amplification willgenerally be linked to a solid support via covalent linkage at their 5′end. Such template strands can be sequenced with the use of “standard”oligonucleotide sequencing primers.

Referring back to the embodiment shown in FIG. 1A, a first sequencingreaction proceeds by sequential addition of nucleotides to the firstfree 3′ hydroxyl group (2). When this sequencing reaction is determinedor deemed to be complete, the strand complementary to the templatestrand (3) is further extended by sequential addition of a known orunknown number of nucleotides to the last nucleotide added in the firstsequencing reaction in a nucleotide addition step. This nucleotideaddition step occurs without sequencing, i.e. nucleotides are addedwithout any determination of the nature of the nucleotide added in eachaddition. Thus, the nucleotides added in the addition step need not bemodified to permit sequencing and are preferably “unmodified”, meaningthat they do not contain any labels required for sequencing, e.g.fluorescent labels, or any blocking groups. It is most preferred to usenaturally occurring nucleotides in this addition step, althoughnucleotides bearing “modifications” other than labels or blocking groupscould be used as long as the modification does not interfere withincorporation into polynucleotide by the chosen polymerase or additionof subsequent nucleotides (e.g. no chain termination effect) to amaterial extent.

The free 3′ hydroxyl group of the last nucleotide added in thenucleotide addition step forms the second free 3′ hydroxyl group(9)—shown in FIG. 1B. The template strand (3) now overhangs the secondfree 3′ hydroxyl group to define a second region of the template to besequenced (8). Thus, in this embodiment the first and second templatestrands are provided by a single template strand (3). A secondsequencing reaction can then proceed from the second free 3′ hydroxylgroup.

The addition of further nucleotides following the first sequencingreaction serves to move the sequencing reaction on to a new position onthe template. Constraints in the sequencing methodology may limit thenumber of sequential base additions which can be made with accuratedetermination of the added nucleotide, meaning that it would not bepossible to reach this point in the template in a single continuoussequencing reaction. By switching to addition of further (unmodified)nucleotides without sequencing and then initiating a second sequencingreaction it is possible to sequence a region farther into the template,by carrying out two sequencing reactions separated by a number of“unsequenced” nucleotides.

The number of further nucleotides added between the first and secondsequencing reactions is typically at least 50, more preferably at least100 and more preferably at least 500.

FIG. 2 illustrates a further embodiment of the invention which issubstantially similar to the embodiment shown in FIG. 1 and describedabove, except that the nucleotides added to further extend thecomplementary strand after completion of the first sequencing reactioncomprise a mixture of unmodified deoxynucleotides (dATP, dCTP, dTTP anddGTP) and a small quantity of deoxyuracil, resulting in incorporation ofuracil into the complementary strand at some of the positions oppositean A in the template strand. An enzyme which cleaves DNA immediatelyadjacent to uracil, for example uracil DNA glycosylase, also known asglycosidase or uracil N-glycosylase or uracil DNA N-glycosylase, maythen be used to generated a second free 3′ hydroxyl group (9) bycleavage of the complementary strand (10).

FIG. 3 illustrates a still further embodiment of the invention whichallows for sequencing of two regions on complementary strands of adouble-stranded template. As shown in FIG. 3A, formation of the startingpolynucleotide template and sequencing of a first region of the template(7) proceeds as described for the embodiment shown in FIG. 1. After thefirst sequencing reaction is deemed to be complete further nucleotidesare added to the 3′ hydroxyl group of the last nucleotide added in thefirst sequencing reaction in order to fully extend the complementarystrand (10) to the full length of the first template strand (3). Thisextension reaction proceeds without sequencing, i.e. withoutdetermination of the nature of the added nucleotides, and results in theproduction of a blunt-ended fully double-stranded template ligated atone end to a hairpin linker (4), which itself is linked to a solidsupport (5) (shown FIG. 3B).

The open end of the double-stranded template (distal from the solidsupport) is then ligated to a second hairpin linker polynucleotide (11)which has a phosphate group at the 5′ end and a free hydroxyl group atthe 3′ end, such that the 5′ end of the second hairpin linker is joinedto the free 3′ end of the extended complementary strand (10). Asillustrated in FIG. 3, the 5′ end of the first template strand isdephosphorylated (or blocked by some other means) such that it is notcapable of forming a phosphodiester linkage with the hydroxyl group atthe 3′ end of the second hairpin linker. The resulting product has anicked circular structure (3C). A substantially equivalent structure mayalso be formed by ligating the second hairpin linker to both the firstand second template strands to form a closed circle and then creating anick in the first template strand (3) or the hairpin linker. The free 3′hydroxyl group (9) of the second hairpin linker provides the second free3′ hydroxyl group for initiation of the second sequencing reaction. Thesecond region of the template to be sequenced (8) is on thecomplementary strand (10), thus this embodiment allows sequencing of tworegions (7, 8) on complementary strands of a double-stranded template.The second free 3′ hydroxyl group is formed in continuous covalentlinkage with the last nucleotide added in the first sequencing reaction,thus there is no need for hybridisation of a separate sequencing primerin order to initiate sequencing of the second region of the template andno need for any fore-knowledge of the sequence of the template.

The second sequencing reaction can be carried out starting from thenicked circle structure shown in FIG. 3C with the use of a stranddisplacing polymerase enzyme. In an alternative embodiment, illustratedin FIGS. 3D and 3E, a portion of what was the first template strand (3)may be removed prior to the second sequencing reaction. This may beachieved, for example, by creating a second nick (12) towards theproximal end of the first template strand near the solid support (5).The nick may be created in the first template strand or within the firsthairpin linker (4), provided that this does not affect the linkage tothe solid support. In a preferred embodiment the nick may be createdwith the use of an appropriate nicking (single side cutting) restrictionenzyme (endonuclease). In order to direct nicking at an appropriateposition the first hairpin linker may contain a recognition sequence fora nicking enzyme which directs cleavage of a single strand at a sitebefore, at or beyond the 3′ end of the first linker. By including thisrecognition site in the hairpin linker, generation of the “nick” isindependent of the sequence of the template and dependent only onligation of the linker to the template. This means that the method maybe universally applied to templates of unknown, and different, sequence.It will be appreciated that nicking can also be accomplished by blockingone side of a standard restriction enzyme cleavage site (in the hairpinlinker sequence or sequences) using methods familiar to those skilled inthe art, e.g., by using thiophosphate linkages in one side of therestriction enzyme recognition site, to prevent cutting in that side,but not in the other, or by the use of DNA methylation.

Following the nicking reaction the resulting product may be denatured inorder to remove the “free” portion of the first template strand locatedupstream (5′) of the cleavage site, as shown in FIG. 3E.

FIG. 4 illustrates a further embodiment of the invention which allowssequencing of two regions on complementary strands of a double-strandedtemplate. This method starts with provision of a fully double-stranded(i.e. blunt ended) polynucleotide template in solution comprising afirst template strand (3) annealed to a complementary strand (10). Thefirst template strand has a 5′ phosphate group, whereas thecomplementary strand lacks a 5′ phosphate group (or 5′ phosphate may beblocked) such that it cannot be joined in phosphodiester linkage to the3′ hydroxyl group of a further nucleotide.

The double-stranded template can be any double-stranded polynucleotidethat it is desired to sequence. In a preferred embodiment it will be afragment of genomic DNA. Double-stranded templates bearing a 5′phosphate on one strand but lacking a 5′ phosphate on the complementarystrand can be generated in a number of different ways. For example, inone embodiment templates can be generated by limited or controlleddigestion of double-stranded DNA fragments bearing 5′ phosphates on bothstrands with a phosphatase enzyme, in order to generate a maximalproportion of strands in which 5′ phosphate is removed from one strandonly.

In a further embodiment double-stranded DNA fragments may be treatedwith phosphatase in order to remove 5′ phosphate from both strands. Theresulting dephosphorylated product may then be subject to limited orcontrolled treatment with a kinase in order to restore 5′ phosphate,limited treatment ensures that a maximal number of fragments will have5′ phosphate restored on one strand only.

In a still further embodiment an adapter, consisting of pairedoligonucleotides having hydroxyl groups at both 3′ and 5′ends of bothstrands may be ligated to one end only of a double-stranded DNA fragmentwhich has 5′ phosphate on both strands. A suitable site for attachmentof the adapter to one end only may be generated by cutting the templatewith a suitable restriction enzyme at a site proximal to the chosen end.

In a still further embodiment an adapter oligonucleotide having a 5′phosphate group may be ligated to one end only of a double-stranded DNAtemplate which has been treated with phosphatase to remove all 5′phosphates. Again, suitable site for attachment of the adapter to oneend of the template may be generated by cutting the template with asuitable restriction enzyme at a site proximal to the chosen end.

A second hairpin linker polynucleotide (11) is ligated to one end of thedouble-stranded template. This second linker has hydroxyl groups at bothits 3′ and 5′ends, thus in the ligation reaction the 3′ end of thesecond linker (OH) is joined to the 5′ end of the first template strand(phosphorylated), but the 5′ end of the second linker (also OH) remainsfree. The second hairpin linker may include a marker moiety, for examplebiotin, which facilitates selection of template molecules that have beensuccessfully ligated to the second linker.

A first hairpin linker (4) is ligated to the second end of thedouble-stranded template. This first linker has a hydroxyl group at the3′ end and a phosphate group at the 5′ end. Thus, in the ligationreaction the 5′ end of the first linker (phosphate) is joined to the 3′end of the first template strand (OH) but the 3′ end of the first linker(OH) remains free. The first hairpin linker may be attached to a solidsupport (5) via a suitable linkage (6). Attachment to the solid supportis preferably carried out after the first linker (4) has been ligated tothe double-stranded template. This provides a means of selection, sinceonly templates that have been correctly ligated to the first linker willbe linked to the solid support.

The terms “first” and “second” hairpin linker are used for conveniencesolely for the purposes of identifying the different structure/functionof the two linkers. The designations “first” and “second” do not requirethe linkers to be joined to the template strands in any particularorder. In the embodiment shown in FIG. 4 it may be convenient for thesecond linker to be ligated to the template before the first linker,since the presence of biotin, or other suitable selectable markermoiety, will enable selection of templates bound to this linker prior tolinkage to the solid support.

The product resulting from ligation of the first and second hairpinlinkers is a closed circle which is “nicked” in two places, asillustrated in FIG. 4A. The “free” strand between the two nicks (10) maybe removed by denaturation to give the structure illustrated in FIG. 4B.Following denaturation the 5′ end (17) of the second linker (11) may betreated with a kinase to restore the 5′ phosphate group.

The 3′ end of the first linker provides the first free 3′ hydroxyl group(2) for initiation of the first sequencing reaction (illustrated FIG.4B). When the first sequencing reaction is deemed to be complete,further nucleotides are added to the 3′ hydroxyl group of the lastnucleotide added in the first sequencing reaction in order to fullyextend the complementary strand (10) to the full length of the firsttemplate strand. This extension reaction proceeds without sequencing,i.e. without determination of the nature of the added nucleotides. The3′hydroxyl end of this extended strand (which will subsequently form thesecond template strand) is ligated to the phosphate group newly added tothe 5′ end of the second hairpin linker (11). Addition of phosphate tothe 5′ end of second linker could be carried out after extension of thecomplementary strand, immediately prior to the ligation reaction, ifconvenient. The product of this ligation is a fully closed circlestructure, as illustrated in FIG. 4C.

In order to generate a second free 3′ hydroxyl group for initiation ofthe second sequencing reaction the closed circle structure shown in FIG.4C may be nicked at a suitable position in the first template strand(3). The position of this first “nick” (13) will determine the positionof the second region to be sequenced on the complementary strand (10,now referred to as the second template strand). Again, the nick may begenerated with the use of a suitable nicking or side-cutter enzyme. Thesecond hairpin linker (11) may include a recognition site for a nickingenzyme which directs nicking/cleavage at a site before, at or beyond the3′ end of the second linker. Thus, generation of the nick (13) isdependent only on the presence of the hairpin linker and is independentof the sequence of the template.

Following generation of a first nick (13), the second sequencingreaction can proceed from the second free 3′ hydroxyl group generated asa result of the nicking reaction with the use of a strand displacingpolymerase enzyme. Alternatively, a portion of the first template stranddownstream from (3′ of) this first nick can be removed altogether priorto the second sequencing reaction. This can be achieved by generating asecond nick (14) spaced apart from (downstream of) the first nick (13).Again a suitable nicking enzyme can be used for this purpose. In orderto form the second nick the first hairpin linker (4) may include arecognition site for a nicking enzyme which directs cleavage at a sitebefore, at or beyond the 5′ end of the first hairpin linker. Followinggeneration of the second nick, the portion of the first template strandlocated between the two nicks may be removed by denaturation, leavingthe structure illustrated in FIG. 4E. The second sequencing reaction maythen proceed from the second free 3′ hydroxyl group (9).

FIG. 5 illustrates a further embodiment of the invention which allowssequencing of two regions on complementary strands of a double-strandedtemplate. The starting template for this method is a partiallyself-complementary covalently closed circle structure as illustrated inFIG. 5A. This structure can easily be formed by ligation of first (4)and second (11) hairpin linkers to the ends of a fully double-stranded,blunt ended template comprising first (3) and second (10) templatestrands.

A first free 3′ hydroxyl group is generated by cleavage of one templatestrand (10) of the covalently closed circle to generate a first “nick”(13) with the use of an appropriate nicking or side-cutter enzyme.Formation of this nick can be directed by including a suitablerecognition site in the first hairpin linker (4) which directs cleavage(nicking) at a site before, at or beyond the 3′ end of the linker. Thisfirst “nick” generates the first free 3′ hydroxyl group (2) forinitiation of the first sequencing reaction. Sequencing may then proceedfrom this initiation point using a strand displacement polymeraseenzyme.

Once this first sequencing reaction is deemed to be complete a secondnick (14) may be generated within the opposite (complementary) templatestrand (3). Formation of this nick can be achieved by the inclusion of asuitable recognition site in the second hairpin linker (11) whichdirects cleavage (nicking) at a site before, at or beyond the 3′ end ofthis linker. This second nick generates the second free 3′ hydroxylgroup (9) for initiation of the second sequencing reaction. Sequencingmay then proceed from this initiation point using a strand displacementpolymerase enzyme. Thus, this method allows sequencing of two targetregions (7,8) on opposite strands of a double-stranded template.

In this method the nucleotides which will ultimately provide the firstand second free 3′ hydroxyl groups are in continuous covalent linkagebefore initiation of the first sequencing reaction through the formationof the closed circle structure illustrated in FIG. 5A. Thus, thisembodiment provides the same technical advantages as the otherembodiments of the invention, since it permits sequencing of twodistinct regions of a template of unknown sequence, without the need forhybridisation of two (or more) separate sequencing primers to provideinitiation points for sequencing.

FIG. 6 illustrates a further embodiment of the invention which allowssequencing of two regions on complementary strands of a double-strandedtemplate. The starting template for this method is a partiallyself-complementary covalently closed circle structure (6A) identical tothat illustrated in FIG. 5A. The covalently closed circle may be formedby ligating first (4) and second (11) hairpin linkers to adouble-stranded polynucleotide comprising first (3) and second (10)template strands.

The second template strand (10) is then “nicked” at two sites (13,14)spaced apart from each other. These nicks can be formed by the inclusionof suitable recognition sequences in the first (4) and second (11)hairpin linkers. The portion of the template strand (10) located betweenthe two nicks is then removed by denaturation to generate the structureshown in FIG. 6C. A first sequencing reaction may then proceed from thefirst free 3′ hydroxyl group (2).

When the first sequencing reaction is complete, the method continuessubstantially as described above with reference to FIG. 4, fromcompletion of the first sequencing reaction. In brief summary, thecovalently closed structure is re-generated by extending the strandformed in the first sequencing reaction to produce a fullydouble-stranded template and then ligating the 3′ end of this extendedstrand to the free 5′ end of the second linker (11). A further nick maythen be formed in the first template strand (3) at a third nicking site(15). Again formation of this nick can be directed by including asuitable recognition sequence in the second linker (11) which directscleavage before, at or beyond its 3′ end. The free 3′ hydroxyl groupgenerated as a result of this nicking then provides the second free 3′hydroxyl group (9) for initiation of the second sequencing reaction.

The second sequencing reaction may be carried out with the use of astrand displacement polymerase. Alternatively, a portion of the stranddownstream of the third nicking site (15) may be removed by nicking thisstrand at a still further (fourth) nicking site (16). Again formation ofthis nick can be directed by including a suitable recognition sequencein the first hairpin linker (4) which directs cleavage before, at orbeyond its 5′ end. The “free” portion of the template strand (3) locatedbetween the two nicking sites (15,16) may then be removed bydenaturation and sequencing can proceed from the second free 3′ hydroxylgroup (9). Thus, this embodiment results in sequencing of two regions(7,8) located on opposite strands of a double-stranded template.

The following preferred features apply mutatis mutandis to allembodiments of the invention:

The polynucleotide template to be sequenced using the method of theinvention may be any polynucleotide that it is desired to sequence. Akey advantage of the invention is that the template may be of unknownsequence. However, the method may also be used to sequence templates ofknown or partially known sequence, for example in re-sequencingapplications. With the use of arrays it is possible to sequence multipletemplates of the same or different sequence in parallel. A particularlypreferred application of the method is in the sequencing of fragments ofgenomic DNA.

Certain embodiments of the method of the invention make use of hairpinlinker polynucleotides, as described above. Self-complementary hairpinpolynucleotide linkers generally have a “stem-loop” structure, formed bybase-pairing of complementary polynucleotides that are covalently linkedat one end. The covalent linkage may be provided by a shortsingle-stranded polynucleotide loop, or may be a non-polynucleotidechemical linkage. Preferred chemical linkages include an arrangement oftwo hexaethylene glycol (heg) spacers separated by an aminodeoxy-thymidine nucleotide. Preferred hairpin linker polynucleotides arethose described in the applicant's published International applicationWO 01/57258. The hairpin linker is effectively “self-priming”, includinga free 3′ hydroxyl group to which further nucleotides may be added. This3′ hydroxyl group provides an initiation point for sequencing of anypolynucleotide template strand ligated to the 5′ end of the hairpin,independent of the sequence of the template strand. The precisenucleotide sequence of the hairpin linker is not material to theinvention.

Hairpin linkers can also provide a means of linking the template strandsto be sequenced to a solid support, for example to form an array. Forexample, hairpins including the above-described linkage consisting oftwo hexaethylene glycol (heg) spacers separated by an aminodeoxy-thymidine nucleotide can be linked to epoxide-modified surfaces(e.g. as described in WO 01/57248). The solid support can be of anysuitable material, for example glass, ceramic, silicon, plastics orother polymeric material. Suitable support materials are also describedin the applicant's published International applications WO 00/06770. Thesupport may take any convenient form. For example it may besubstantially planar, such as a slide or “DNA chip”, or may be a threedimensional support, or a support formed from multiple discrete units,for example beads of glass, plastics, magnetic material etc.

Templates may be attached to a solid support via any suitable linkagemethod known in the art. Preferably linkage will be via covalentattachment. The templates may be attached to the support directly or viaa hairpin linker. If hairpin linkers are used then they may be attachedto the solid support before they are linked to template(s) to besequenced. For example, hairpin linkers may be attached to the solidsupport to form an array prior to attachment of the template(s).Techniques for forming arrays of hairpin linkers are described in WO01/57248. The templates/linkers are preferably attached to the solidsupport via a covalent linkage. Linkage may be made via any part of thetemplate or hairpin, provided that this does not interfere with theability of the template/linker to participate in a sequencing reaction.If hairpins are used, the linkage may advantageously be made via aninternal portion of the hairpin, leaving the 3′ end free to initiate asequencing reaction and the 5′ end free for attachment to the template.In the case of template strands generated by solid-phase amplificationattachment to the solid support will usually be via covalent linkagewith the 5′ end of the template.

If the templates are “arrayed” on a solid support then the array maytake any convenient form. An advantage of the method of the invention isthat large numbers of templates of different sequence can be processedin parallel, since the method is independent of the sequence of thetemplate and does not require any sequence-specific hybridisation stepsto take place on the array. Thus, the method is applicable to all typesof “high density” arrays, including single-molecule arrays and clusteredarrays.

Single molecule arrays are formed by immobilisation of a singlepolynucleotide molecule at each discrete site that is detectable on thearray. Single-molecule arrays comprised of nucleic acid molecules thatare individually resolvable by optical means and the use of such arraysin sequencing are described, for example, in WO 00/06770. Singlemolecule arrays comprised of individually resolvable nucleic acidmolecules including a hairpin loop structure are described in WO01/57248. The method of the invention is suitable for sequencingtemplate molecules on single molecule arrays prepared according to thedisclosures of WO 00/06770 of WO 01/57248.

However, it is to be understood that the scope of the invention is notintended to be limited to the use of the method in connection withsingle molecule arrays. The method may be used for sequencing onessentially any type of array formed by immobilisation of nucleic acidmolecules on a solid support, and more particularly any type ofhigh-density array. In addition to single molecule arrays suitablearrays may include, for example, multi-polynucleotide or clusteredarrays in which distinct regions on the array comprise multiple copiesof one individual polynucleotide molecule or even multiple copies of asmall number of different polynucleotide molecules (e.g. multiple copiesof two complementary nucleic acid strands).

In particular, the method of the invention may be utilised forsequencing on multi polynucleotide or “clustered” arrays.Multi-polynucleotide or clustered arrays of nucleic acid molecules maybe produced using techniques generally known in the art. By way ofexample, WO 98/44151 and WO 00/18957 both describe methods of nucleicacid amplification which allow amplification products to be immobilisedon a solid support in order to form arrays comprised of clusters or“colonies” of immobilised nucleic acid molecules. The nucleic acidmolecules present on the clustered arrays prepared according to thesemethods may be suitable templates for sequencing using the method of theinvention. However, the invention is not intended to be limited to useof the method in sequencing reactions carried out on clustered arraysprepared according to these specific methods.

When using arrays formed from nucleic acid “colonies”, such as arraysprepared according to the teaching in WO 98/44151 and WO 00/18957, it isnot necessary to use a hairpin linker polynucleotide to link thetemplate molecule to the array and provide the first free 3′ hydroxylgroup for sequencing. The arrays of WO 98/44151 and WO 00/18957 areformed by “on-chip” amplification using primers and templatesimmobilised on a solid support. Once amplification is complete, thecolonies may comprise pluralities of double-stranded nucleic acidmolecules. These double-stranded molecules may be processed to formtemplates suitable for use in the methods of the invention, either byforming a “nick” in one strand only of the double-stranded molecules orby removing a portion of one strand altogether. Either way, a free 3′hydroxyl group will be formed on one strand which serves as aninitiation point for the first sequencing reaction. After the firstsequencing reaction is complete, formation of the second free 3′hydroxyl group can proceed using some of the alternatives describedabove, with appropriate modification.

As outlined above, sequencing can be carried out using any suitable“sequencing-by-synthesis” technique, wherein nucleotides are addedsuccessively to a free 3′ hydroxyl group, resulting in synthesis of apolynucleotide chain in the 5′ to 3′ direction and the nature of thenucleotide added is preferably determined after each addition.

One preferred sequencing method which can be used in the methods of theinvention relies on the use of modified nucleotides that can act aschain terminators. Once the modified nucleotide has been incorporatedinto the growing polynucleotide chain complementary to the region of thetemplate being sequenced there is no free 3′-OH group available todirect further sequence extension and therefore the polymerase can notadd further nucleotides. Once the nature of the base incorporated intothe growing chain has been determined, the 3′ block may be removed toallow addition of the next successive nucleotide. By ordering theproducts derived using these modified nucleotides it is possible todeduce the DNA sequence of the DNA template. Such reactions can be donein a single experiment if each of the modified nucleotides has attacheda different label, known to correspond to the particular base, tofacilitate discrimination between the bases added at each incorporationstep. Alternatively, a separate reaction may be carried out containingeach of the modified nucleotides separately.

The modified nucleotides carry a label to facilitate their detection.Preferably this is a fluorescent label. Each nucleotide type may carry adifferent fluorescent label. However the detectable label need not be afluorescent label. Any label can be used which allows the detection ofthe incorporation of the nucleotide into the DNA sequence.

One method for detecting the fluorescently labelled nucleotidescomprises using laser light of a wavelength specific for the labellednucleotides, or the use of other suitable sources of illumination. Thefluorescence from the label on the nucleotide may be detected by a CCDcamera or other suitable detection means.

The methods of the invention are not limited to use of the sequencingmethod outlined above, but can be used in conjunction with essentiallyany sequencing methodology which relies on successive incorporation ofnucleotides into a polynucleotide chain. Suitable techniques include,for example, Pyrosequencingm, FISSEQ (fluorescent in situ sequencing),MPSS (massively parallel signature sequencing) and sequencing byligation-based methods.

The invention will be further understood with reference to the followingnon-limiting experimental example:

EXAMPLE Methods and Materials

(1) Extension of Polynucleotide Strands with Polymerase and all FourNucleotides-without Sequencing.

A polymerase (e.g., Taq DNA polymerase, Pfu plymerase, Klenow fragmentof E. coli Pol I, etc.) is added at a suitable concentration in a bufferdesigned to be used with that enzyme. All four deoxynucleotidetriphosphates are added at a concentration in the 1-11 micromolar range.Extension is for a time (10 sec to a few minutes) that will allowapproximately 500 base pairs to be incorporated.

(2) Extension of Polynucleotide Strands with Polymerase, Deoxyuracil andall Four Nucleotides.

As in previous description (3), but with a small amount (1 part in 100,e.g. compared with standard deoxythymidine triphosphate) of deoxyuraciltriphosphate mixed in. The amount of U nucleotide is adjusted to provideincorporation of U at a rate of the order of 1U every 500 nucleotidesadded.

(3) Cleavage of U-Containing DNA with Uracil DNA Glycosylase (UDG).

After an appropriate wash, chips containing DNA can be incubated in 14parts UDG Buffer (70 mM Hepes-KOH pH 8.0, 1 mM dithiothreitol, 1 mMEDTA), 1 part uracil DNA glycosylase (BRL). After e.g. two hours ofincubation at 37° C. the chips are optionally washed with a high pH washsolution and heated to 94° C. for up to 10 min to kill the enzyme. Thisstep may not be necessary for a chip. They are then subjected toappropriate washes in an appropriate buffer and made ready for the nextstep.

(4) On-Chip Ligation.

Hairpin DNA on the surface at a density to allow appropriate singlemolecule array analysis (10⁷-10⁹ molecules per square cm) anddouble-stranded genomic DNA are mixed together. The molecules to beligated must contain the appropriate phosphate or hydroxyl termini asdescribed above. The DNA molecules are incubated in the presence of a1:2 dilution of “2× Quick Ligation Reaction Buffer” (NEB=New EnglandBiolabs, Beverly, Mass.) and a 1:20 dilution of NEB “Quick T4 DNALigase”, both components of the NEB kit called “Quick Ligation Kit”, atroom temperature for approximately 1 h. Subsequently, washing isperformed in a suitable wash reagent(s) or buffer(s), using suitabletimes and temperatures.

(5) Nicking Reaction.

Nicking of DNA on a surface via specific recognition sequences in theknown part of the DNA is described here. The surface equivalent ofroughly a solution amount of 1.25 pmoles of DNA is digested at 55° C.for 30 minutes with N.BstNBI (50 Units/mL final concentration) in itssupplied buffer (NEBuffer N.BstNBI at “1×” final concentration) from themanufacturer (NEB). This experiment can also be performed with littlemodification in a flow-cell where the substrate comprises DNA ligated toDNA hairpins that are covalently attached to the glass surface of theflow-cell. In this case, the attachment of the DNA to a solid support,the glass, obviates the need to employ a DNA purification step betweenenzyme steps; instead, products can be removed and new reagents added byflowing solutions through the cell. Suitable wash buffers as known inthe art can be employed.

It will be appreciated that nicking can also be accomplished by blockingone side of a standard restriction enzyme cleavage site (in the hairpinoligonucleotide sequence or sequences) using methods familiar to thoseskilled in the art, e.g., by using thiophosphate linkages in one side ofthe restriction enzyme's recognition site, to prevent cutting in thatside, but not in the other.

(6) Strand-Displacement Polymerisation.

Strand-displacement polymerisation is carried out using a suitablestrand-displacement polymerase (e.g., BST Polymerase). The reactionconditions are, e.g., “1× Thermopol buffer” (final concentrations: 20 mMTris-HCl pH 8.8 measured @25° C., 10 mM KCl, 10 mM (NH₄)₂SO₄, 2 mMMgSO₄, 0.1% Triton X-100), all four dNTPs at a concentration of between1 and 200 micromolar, 160 Units/mL (final concentration) of BST largefragment polymerase (NEB). Reactions are performed, for 2 hrs. at 60°C., for example, followed by washing in a suitable wash buffer, usingsuitable times and temperatures.

(7) Denaturation.

Denaturation may be performed by any of the methods known to thoseskilled in the art. Typically there are three preferred methods:

-   -   Chips are washed in very low ionic strength or distilled water.    -   Chips are incubated at high temperature (80-90° C., typically)        for a few minutes in an appropriate buffer or at 40-70° C. in        50% deionized formamide dissolved in water.    -   Chips are incubated at high pH briefly, i.e. pH greater than 10.        This latter method is least preferred as it may be damaging to        the surface chemistry of the chip.

(8) Phosphatasing and Kinasing.

See e.g., Maniatis, T., E. F. Fritsch and J. Sambrook. 1982. MolecularCloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.

1. (canceled)
 2. A method for pairwise sequencing of a polynucleotidetemplate, the method comprising: (a) providing a polynucleotide templateimmobilized on a surface, said polynucleotide template comprising afirst template strand having a first region and a 5′ phosphate group,and a first self-complementary hairpin polynucleotide linker comprisinga loop region and a stem region, wherein the 5′ end of one strand of thestem region is linked to the 3′ end of said first template strand andthe 3′ end of the other strand of the stem region comprises a first free3′ hydroxyl group used for initiating sequencing of the first region ofthe first template strand, (b) performing a firstsequencing-by-synthesis reaction comprising sequential incorporation ofdifferent complementary reversibly-terminated nucleotides into the 3′end of said first self-complementary hairpin, thereby determining thesequence of the first region of the first template strand, wherein eachof said nucleotides comprises a fluorescent label and a 3′ blockinggroup, the blocking group prevents any further nucleotide incorporationinto the 3′ end of the polynucleotide template, and wherein saidsequential incorporation comprise (i) incorporating one of saidnucleotides into the first free 3′ hydroxyl group and detecting afluorescent signal generated from the fluorescent label using a CCDcamera or other fluorescence detection means, and (ii) cleaving thefluorescent label and the 3′ blocking group from said one of saidnucleotides in the 3′ end of said polynucleotide template, therebyyielding a free 3′ hydroxyl group before another of said nucleotides isincorporated into the 3′ end of the polynucleotide template, (c) addingan unlabeled nucleotide to the free 3′ hydroxyl group in the lastnucleotide added in step (b) and performing an extension reaction in thepresence of different unlabeled nucleotides, thereby generating a secondtemplate strand having a free 3′ hydroxyl group, wherein the secondtemplate strand is complementary to the full length of said firsttemplate strand and comprises a second region, (d) ligating a secondself-complementary hairpin polynucleotide linker to the free 3′ hydroxylgroup of the second template strand and the 5′ phosphate group of thefirst template strand, thereby generating a self-complementary, circularpolynucleotide template, (e) cleaving the circular polynucleotidetemplate, thereby yielding a 3′ end comprising a second free 3′ hydroxylgroup used for initiating sequencing of the second region of the secondtemplate strand, (f) performing a second sequencing-by-synthesisreaction comprising sequential incorporation of different complementaryreversibly-terminated nucleotides into the 3′ end generated in step (e),thereby determining the sequence of the second region of the secondtemplate strand, wherein each of said nucleotides comprises afluorescent label and a 3′ blocking group, the blocking group preventsany further nucleotide incorporation into the 3′ end of thepolynucleotide template, and wherein said sequential incorporation afterstep (e) comprises (i) incorporating one of said nucleotides into thesecond free 3′ hydroxyl group and detecting a fluorescent signalgenerated from the fluorescent label using a CCD camera or otherfluorescence detection means, and (ii) cleaving the fluorescent labeland the 3′ blocking group from said one of said nucleotides in the 3′end of said polynucleotide template, thereby yielding a free 3′ hydroxylgroup before another of said nucleotides is incorporated into the 3′ endof said polynucleotide template; wherein said first template strandranges in length from 100 nucleotides to 1 kb.
 3. The method of claim 2,wherein the cleaving the circular polynucleotide template in step (e)comprises contacting the circular polynucleotide template with a nickingendonuclease.
 4. The method of claim 3, wherein the secondself-complementary hairpin polynucleotide linker comprises a recognitionsite for a nicking endonuclease which directs cleavage by the nickingendonuclease at a site before, at or beyond the 3′ end of the secondself-complementary hairpin polynucleotide linker.
 5. The method of claim2, wherein the second sequencing-by-synthesis reaction of step (f) iscarried out using a strand-displacing polymerase enzyme.
 6. The methodof claim 2, wherein cleaving the circular polynucleotide template instep (e) comprises contacting the circular polynucleotide template withfirst and second nicking endonucleases.
 7. The method of claim 6,wherein the first self-complementary hairpin polynucleotide linkercomprises a recognition site for a first nicking endonuclease whichdirects cleavage by the nicking endonuclease at a site before, at orbeyond the 5′ end of the first self-complementary hairpin linkingpolynucleotide, and wherein the second self-complementary hairpinpolynucleotide linker comprises a recognition site for a second nickingendonuclease which directs cleavage by the nicking endonuclease at asite before, at or beyond the 3′ end of the second self-complementaryhairpin polynucleotide linker.
 8. The method of claim 6 wherein,subsequent to cleaving the circular polynucleotide template in step (e)but prior to the second sequencing-by-synthesis reaction of step (f), aportion of the first template strand is removed by denaturation.
 9. Themethod of claim 2, wherein the polynucleotide template forms part of anarray.
 10. The method of claim 2, wherein the firstsequencing-by-synthesis reaction of step (b) comprises incorporation of10 to 200 consecutive nucleotides.
 11. The method of claim 2, whereinthe second sequencing-by-synthesis reaction of step (f) comprisesincorporation of 10 to 200 consecutive nucleotides.
 12. The method ofclaim 2, wherein the extension reaction of step (c) comprisesincorporation of at least 100 consecutive nucleotides.
 13. The method ofclaim 2, wherein the extension reaction of step (c) comprisesincorporation of at least 200 consecutive nucleotides.
 14. The method ofclaim 2, wherein the polynucleotide template is directly linked to thesurface of a solid support.
 15. The method of claim 2, wherein theextension reaction of step (c) is performed without determining theidentities of the unlabeled nucleotides added in step (c).